Method for the manufacture of nerve regeneration-inducing tube

ABSTRACT

A method for manufacturing a nerve regeneration-inducing tube with excellent pressure resistance, shape recovery property, anti-kink property, film exfoliation resistance, resistance to invasion of outer tissues, and leakage resistance. The tubular body is formed by weaving together fibers made up of biodegradable polymer. The outer surface of the tubular body is coated multiple times with a collagen solution. The lumen of the tubular body is filled with collagen. Viscosity of the collagen solution that is first applied to the outer surface of the tubular body is between 2 to 800 cps. Viscosity of the collagen solution that is subsequently applied is higher than viscosity of the first applied collagen solution.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.12/744,179, filed May 21, 2010, and wherein application Ser. No.12/744,179 is a national stage application filed under 35 USC §371 ofInternational Application No. PCT/JP2008/072038, filed Dec. 4, 2008, andwhich is based upon and claims the benefit of priority from the priorJapanese Patent Application No. 2007-317462, filed on Dec. 7, 2007, theentire contents of which are incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method for the manufacture of a nerveregeneration-inducing tube by which peripheral nerve cut or excised byaccident or surgical operation is reconnected utilizing the elongationof nerve cells. More particularly, the present relates to a method whereclose adhesion of a tubular body comprising a biodegradable polymerconstituting the nerve regeneration-inducing tube with collagen appliedon the outer surface of the tubular body is enhanced whereby the initialstrength, flexibility, etc. of the entire nerve regeneration-inducingtube are improved.

BACKGROUND ART

There are many examples where damage of peripheral nerve caused byaccident or the like is unable to be completely restored. There are alsomany clinical examples where peripheral nerve must be excised as aresult of surgical operations in general. In the damage of peripheralnerves, autologous nerve grafting has been an only means besides adirect anastomosis. However, the result thereof is not alwayssatisfactory but recovery of sensory perception and capacity forlocomotion are bad and the aftereffect due to erroneous governing isnoted as well. In addition, there are many patients complaining not onlythe aftereffect such as pain and deficiency in sensory perception butalso the abnormal sensory perception of the diseased area or,particularly, pain.

An attempt for the regeneration of nerve by connection of gaps ofperipheral nerve using a connecting tube made of artificial materialshas been briskly carried out since early 1980's. However, all of thestudies of connecting channels using non-absorptive synthetic artificialmaterials have resulted in failure. In order to solve the above, it isnecessary to consider in the followings such as that invasion ofconnective tissues from outside is prevented during the regeneration ofnerve bundles, that substance interchange inside and outside thechannels or neogenesis of capillary blood vessels in channel walls isnecessary, that a substance acting as a scaffold suitable for the growthof Schwann cells and axon in the channel is necessary and that, afterthe regeneration, the used material is degraded and absorbed. Takingthose conditions into consideration, studies for artificial nerveconnecting tube by a biodegradable and absorbable material have beencarried out thereafter.

With regard to the regeneration of peripheral nerve, attempts forextending the distance between the stumps which are able to beregenerated using a silicone tube have been conducted since a siliconetube model was reported in 1982. However, since nutrients are unable topermeate through the wall of silicone tube, there is a problem such asthat the nutrients are not sufficiently provided to nerve axon wherebycapillary blood vessel is unable to be produced in silicone and nosatisfactory nerve regeneration has been available even when a siliconetube is used. Further, even if the nerve is able to be regenerated,there is a problem that the silicone tube which is a foreign substanceanyway must be removed by means of further surgical operation, etc.

On the other hand, regeneration of peripheral nerve using a tubecomprising a biodegradable polymer in place of a silicone tube has beenattempted. When a nerve regeneration tube comprising a biodegradablepolymer is used, the nerve regeneration tube is gradually degraded andabsorbed in vivo by hydrolysis or by the action of enzymes after thenerve is regenerated whereby there is no need of taking out it by ameans such as further surgical operation.

With regard to a nerve regeneration tube comprising a biodegradablepolymer as such, there is a disclosure in, for example the PatentDocument 1, for an auxiliary material for nerve regeneration whichcomprises bundles of collagen fiber on which laminin and fibronectin arecoated. In the Patent Document 2, there is a disclosure for anartificial nerve tube which comprises a tubular body comprisingbiodegradable and absorbable material and, in the lumen of the tubularbody, a collagen body having gaps and penetrating the tubular bodynearly in parallel to the axial line of said tubular body where the gapis filled with a matrix gel containing collagen, laminin, etc. In thePatent Document 3, there is a disclosure for an artificial nerve tubewhich comprises a tubular body comprising biodegradable and absorbablematerial and laminin-coated collagen fiber bundles inserted into thelumen of the tubular body nearly in parallel to the axial line of thetubular body. In the Patent Document 4, there is a disclosure for asubstrate material for the reconstruction of nerves having a structurewhere fibers comprising a bioabsorbable material are bundled. In thePatent Document 5, there is a disclosure for a support such as sponge,tube or coil comprising collagen. In the Patent Document 6, there is adisclosure for a support which is composed of a spongy fine matrixcomprising a biodegradable material or a bioabsorbable material and alinear biotissue induction path or a linear organ induction path. In thePatent Document 7, there is a disclosure for a nerve regeneration tubecontaining a sponge comprising a biodegradable polymer material and areinforcing material comprising a biodegradable polymer having longerperiod for degradation and absorption than that of said sponge whereinthe inner side thereof comprises sponge.

The nerve regeneration tubes as such are usually manufactured in such amanner that collagen is applied to the outer surface of the tubular bodyknitted with ultrafine fiber comprising the biodegradable polymer andthen collagen is filled in the inner area of the tubular body. However,since the close adhesion of the collagen applied to the outer surface ofthe tubular body with the biodegradable polymer of the tubular body ispoor, there is a problem in the strength, the flexibility, etc. in itsactual use.

PATENT DOCUMENTS

-   1. Japanese Patent Application Laid-Open (JP-A) No. 237139/93-   2. WO 98/22155-   3. WO 99/63908-   4. Japanese Patent Application Laid-Open (JP-A) No. 2000-325463-   5. Japanese Patent Application Laid-Open (JP-A) No. 2001-70436-   6. Japanese Patent Application Laid-Open (JP-A) No. 2002-320630-   7. Japanese Patent Application Laid-Open (JP-A) No. 2003-19196

BRIEF DESCRIPTIONS OF DRAWINGS

FIG. 1 is an illustrative drawing for the method of evaluation ofpressure resistance.

FIG. 2 is an illustrative drawing for the method of evaluation of shaperecovery property.

FIG. 3 is an illustrative drawing for the method of evaluation ofanti-kink property.

FIG. 4 is an illustrative drawing for the method of evaluation of filmexfoliation resistance.

FIG. 5 shows an SEM image (50×) of the tubular body of Example.

DISCLOSURE OF THE INVENTION Problem that the Invention is to Solve

The present invention has been created in view of the current status ofthe prior art as such and an object of the present invention is toprovide a method for the manufacture of a nerve regeneration-inducingtube where a collagen solution is applied on the outer surface of atubular body knitted with ultrafine fiber comprising a biodegradablepolymer while collagen is filled in the inner area of the tubular body,which nerve regeneration-inducing tube is excellent in pressureresistance, shape recovery property, anti kink property, filmexfoliation resistance, property of prevention of invasion of outertissues and leakage resistance.

Means for Solving the Problem

In order to achieve the object as such, the present inventor hasconducted extensive investigation for a method where close adhesion ofthe tubular body knitted from the biodegradable polymer fiber withcollagen applied onto the outer surface thereof is enhanced and, as aresult, it has been found that a nerve regeneration-inducing tubeexcellent in pressure resistance, shape recovery property, anti-kinkproperty, film exfoliation resistance, property of prevention ofinvasion of outer tissues and leakage resistance is able to bemanufactured in high efficiency when viscosity (or concentration) of thecollagen solution firstly applied to the outer surface of the tubularbody is made low and, in the application thereafter, viscosity (orconcentration) of the collagen solution is made higher than beforewhereupon the present invention has been achieved.

Thus, the present invention is a method for the manufacture of a nerveregeneration-inducing tube in which the outer surface of the tubularbody knitted with a plurality of ultrafine fibers comprisingbiodegradable polymer is coated by application of a collagen solutionfor plural times and then collagen is filled in the inner area of theabove tubular body, characterized in that, viscosity of the collagensolution which is firstly applied on the outer surface of the tubularbody is made 2 cps to 800 cps or, preferably, 5 cps to 200 cps.

In the preferred embodiment of the method of the present invention,viscosity of a collagen solution to be applied later is made high ascompared with that of the firstly-applied one where the viscosity ispreferably made high in two or more stages. Alternatively, a collagensolution in the viscosity of the collagen solution which is firstlyapplied to the outside of the tubular body is applied for plural times.

Furthermore, in the preferred embodiment of the method of the presentinvention, the biodegradable polymer is at least one polymer which isselected from the group consisting of polyglycolic acid, polylactic acidand a lactic acid-caprolactone copolymer. The present invention alsorelates to a nerve regeneration-inducing tube which is characterized inbeing manufactured by the above method.

Advantages of the Invention

In the manufacturing method of the present invention, viscosity (orconcentration) of the collagen solution which is firstly applied to theouter surface of the tubular body knitted with a biodegradable polymerfiber is made low whereby it is now possible to provide a nerveregeneration-inducing tube in which the tubular body comprising thebiodegradable polymer is uniformly and tightly adhered to collagen andwhich is excellent in pressure resistance, shape recovery property,anti-kink property, film exfoliation resistance, property of preventionof invasion of outer tissues and leakage resistance.

Best Mode for Carrying Out the Invention

In the method of the present invention, the nerve regeneration-inducingtube is manufactured in such a manner that a collagen solution isapplied for plural times to outer surface of a tubular body knitted withplural ultrafine fibers comprising a biodegradable polymer to coat andcollagen is further filled in the lumen of the tubular body.

Examples of the biodegradable polymer constituting the tubular bodyinclude polylactic acid, polyglycolic acid, polycaprolactone, a lacticacid-glycolic acid copolymer, a lactic acid-caprolactone copolymer, aglycolic acid-caprolactone copolymer, polydioxanone and glycolicacid-trimethylenecarboxylic acid. In view of easy availability andhandling, it is preferred to use polyglycolic acid, polylactic acid or alactic acid-caprolactone copolymer and it is particularly preferred touse polyglycolic acid. Each of those biodegradable polymers may be usedsolely or two or more thereof may be used by mixing.

In the present invention, diameter of the ultrafine fiber comprising thebiodegradable polymer is preferred to be from 1 to 50 μm. When the fiberdiameter is too small, the fiber gap becomes dense whereby it may happenthat collagen is hardly permeated into the tubular body or thatflexibility of the tubular body lowers. On the contrary, when the fiberdiameter is too large, the retained amount of collagen becomes smallwhereby it may happen that the growing speed of the nerve does not riseor that the strength of the tubular body becomes insufficient. Morepreferably, diameter of the ultrafine fiber is 3 to 40 μm, and furtherpreferably 6 to 30 μm.

In the formation of the tubular body, it is preferred that 5 to 60 ofthe ultrafine fibers comprising the biodegradable polymer and having theabove fiber diameter are bundled and alternately knitted as warps andwoofs. When the numbers of the ultrafine fibers to be bundled are toosmall, it may happen that the strength of the tubular body becomesinsufficient or that a sufficient retained-amount of collagen is unableto be secured. On the contrary, when the numbers of the ultrafine fibersto be bundled are too many, it may happen that a tubular body in finediameter is unable to be prepared or that flexibility of the tubularbody are unable to be secured. More preferably, the numbers of theultrafine fibers are 10 to 50, and further preferably 20 to 40.

When a tubular body is formed by an alternate knitting of the ultrafinefiber bundles, the pore size of the network is preferred to be about 5to 300 μm, and more preferably 10 to 200 μm. When the pore size of thenetwork is too small, it may happen that growth of the cells and thetissues is inhibited due to the lowering of invasion of capillary bloodvessel or due to the lowering of water permeability. When it is morethan about 300 μm, invasion of the tissues becomes excessive wherebygrowth of the cells and the tissues may be inhibited.

It is preferred that although inner diameter and outer diameter of thetubular body are decided to accord with the size of the nerve to beconnected and, when the production cost and the time limitation aretaken into consideration, it is preferred that many kinds of tubularbodies where the sizes are varied are previously prepared. Although thesize of the tubular body depends on the site of the nerve to beregenerated and on the necessary strength, it is usual that the innerdiameter is 0.1 to 20 mm, the outer diameter is 0.15 to 25 mm, the wallthickness is 0.05 to 5 mm and the length is 1.0 to 150 mm. When the wallthickness is too thick, that may obstruct the regeneration of thebiotissues while, when it is too thin, degradation and absorption of thetubular body are too quick whereby the shape may not be held until theregeneration of the nerve finishes. Further, when the inner diameter tothe nerve to be connected is too big, there is a possibility thatelongation of the nerve is unable to be done appropriately.

In the present invention, the outer surface of the tubular body iscoated by applying a collagen solution for several times by a methodwhich has been known among persons skilled in the art while the innerarea (lumen) of the tubular body is filled by charging collagen therein.With regard to the collagen to be used for application to the outersurface of the tubular body and for filling into the inside of thetubular body, there may be used collagen which has been conventionallyused as a scaffold for nerve regeneration. Examples thereof include typeI collagen, type III collagen, and type IV collagen and the like andeach of them may be used solely or plural ones may be used by mixing.With regard to the collagen, it is preferred to use a purified one whereconcentration of sodium chloride contained therein is made 2.0% byweight or less, preferably 0.1 to 1.5% by weight on a dry basis. Thecollagen may also contain laminin, heparan sulfate proteoglycan,entactin and growth factor. Examples of the growth factor include EGF(epidermal growth factor), βFGF (fibroblast growth factor), NGF (nervegrowth factor), PDGF (platelet-derived growth factor), IGF-1(insulin-like growth factor) and TGF-β (transforming growth factor).With regard to the collagen solution, it is preferred that, after everyone application thereof in a form of a solution in hydrochloric acidusing a brush or a writing brush, the solution is completely dried andthen the next application is conducted whereby a plurality ofapplications are done.

The most important feature of the method of the present invention isthat, when the outer surface of the tubular body is applied with acollagen solution, a low-viscosity solution of 2 to 800 cps, preferably5 to 200 cps is used as a collagen solution for the first application.Frequency of the application of this low-viscosity solution is preferredto be from once to ten times, preferably once to five times. As a resultof application of the low-viscosity solution of said range firstly, thecollagen solution is well permeated among the ultrafine fibers of thebiodegradable polymer constituting the tubular body whereby adhesion andunified feel of the biodegradable polymer with collagen is able to besignificantly enhanced. When a high-viscosity solution having higherviscosity than the above is applied firstly, the collagen solution isunable to be permeated among the ultrafine fibers whereby the collagenbecomes a filmy state after drying whereupon there is a risk thatcollagen is exfoliated from the tubular body. When such a nerveregeneration-inducing tube is used, there is resulted inhibition ofinvasion of the blood vessel into the tubular body or inhibition ofgrowth of nerve cells.

In the method of the present invention, it is preferred that, firstly, alow-viscosity collagen solution is applied for several times so as toform a sealing of gap between fibers constituting the tubular body and athin layer and then a collagen solution of higher viscosity of 200 to30,000 cps is applied thereon. That is because, in an application of thelow-viscosity solution only, very many times of application arenecessary for achieving a predetermined thin layer thickness whereby theworking ability is bad. Frequency of the application of thishigh-viscosity solution is desired to be from once to fifty times,preferably once to thirty times. When the frequency of application ofthe high-viscosity solution is too many, that causes a lowering of theshape recovery property and, for example, when a diseased area iscrushed with something after the surgical operation, the strain resultedon the tube is not recovered whereby it may clog the nerve regenerationpath. Further, since collagen has a relatively quick biodegradationspeed, there is little merit even when the application frequency isincreased too much.

Actually, it is preferred that the viscosity of the collagen solution ismade higher in multiple stages of two or more after the firstapplication of the low-viscosity solution. For example, the viscosity ofthe collagen solution to be applied can be raised in three stages of 2to 200 cps, 200 to 3,000 cps and 3,000 to 30,000 cps. In that case,permeation among the ultrafine fibers of the tubular body and formationof film on the surface are conducted by the first low-viscositysolution, adhesion to this film is done using the next medium-viscositysolution to conduct the sealing of the network and the lasthigh-viscosity solution is adhered to this sealed collagen layer toenhance the strength whereby the coating with a strong initial strengthis able to be efficiently carried out. Further, the gap of the viscosityapplied in a stepwise manner as such is made little whereby it ispossible to improve the operating ability of the applying work or toreduce the uneven application or the place left unapplied.

It is preferred that the tubular body where collagen is coated or filledis subjected to freezing, freeze-drying and cross-linking treatments tocross-link the collagen. Preferably, the freezing is carried out underthe condition of −10 to −196° C. for 3 to 48 hours. As a result of thefreezing, fine ice is formed among the collagen molecules and thecollagen solution results in a phase separation to give sponge. Afterthat, the above frozen collagen solution is freeze-dried in vacuopreferably at about −40 to −80° C. and preferably about 12 to 48 hours.As a result of freeze-drying, fine ice among the collagen molecules isevaporated and, at the same time, the collagen sponge becomes fine.Examples of the cross-linking method include γ-ray cross-linking,ultraviolet cross-linking, electronic ray cross-linking, thermaldehydration cross-linking, glutaraldehyde cross-linking, epoxycross-linking and water-soluble carbodiimide cross-linking and, amongthem, a thermal dehydration cross-linking where the cross-linking degreeis able to be easily controlled and living body is not effected even byconducting the cross-linking treatment is preferred. The thermaldehydration cross-linking is conducted in vacuo at, for example, about105 to 150° C., more preferably about 120 to 150° C., and furtherpreferably about 140° C. for about 6 to 24 hours, more preferably about6 to 12 hours, and further preferably about 12 hours. When thecross-linking temperature is too high, there is a possibility that thestrength of the biodegradable and absorbable material lowers while, whenit is too low, there is a possibility that no sufficient cross-linkingreaction takes place.

Since the tubular body comprising the biodegradable polymer and thecollagen are tightly adhered with each other in the nerveregeneration-inducing tube manufactured as mentioned above, the initialstrength and elasticity which are not lower than the sum of the strengthof each are available. To be more specific, in the nerveregeneration-inducing tube of the present invention, the strain rate(pressure resistance) when compression is done by applying the load of100 N/m from the side in the direction of diameter is not more than 15%,preferably 0.1 to 10% and, further, the recovery rate (shape recoveryproperty) in 50% of the strain when similar compression is done so as togenerate of 50% strain of the tube (until the diameter of the tubebecomes one half) is not less than 60%. Pressure resistance is on theassumption of the resistance to the load for the nerveregeneration-inducing tube due to the work by a medical device uponconnection of nerve and to the treatment after the surgical operationand, generally, the more the thickness of the collagen layer, the morethe pressure resistance. However, when the tubular body and collagen arenot tightly adhered with each other but the film is separated, thepressure resistance is not able to be so much expected. In addition, theshape recovery property is on an assumption for a recovery of the shapefrom the strain due to the work by a medical device upon connection ofnerve (such as too strong picking by a pair of tweezers) or the shock tothe diseased area after the surgical operation and, if the shaperecovery property is low, strain remains in the tube and the nervegrowth path is inhibited.

Further, the nerve regeneration-inducing tube of the present inventionhas a limiting curved rate (anti-kink property) of not less than 10% andalso has a high resistance to exfoliation of the film. The limitingcurved rate shows the range where bending is possible without causing akink and is an index concerning the movable region upon connection ofthe nerve. When the limiting curve rate is less than 10%, it is notpossible to use for the case where a curved nerve growth path isnecessary and, even if used, tension is applied to the nerve and thereis a risk of causing the inhibition of growth of the nerve and theinflammation caused by compression of outer tissues. Resistance toexfoliation of the film is the resistance to exfoliation and crack ofthe coated collagen. The reason why collagen is coated on the entireouter surface of the tubular body is to prevent the invasion of outertissue to the nerve growth path (invasion of outer tissues-preventionproperty) and to prevent the leakage of the collagen sponge in the innerarea of the tubular body to outside (leakage resistance) and, when thecoated collagen is exfoliated or cracked, there is a risk that the aboveproperties are unable to be secured. In the nerve regeneration-inducingtube of the present invention, the tubular body and collagen are tightlyadhered with each other and there is no separated film whereby a highanti-kink property is able to be achieved and, at the same time, thereis no possibility that the exfoliation and the crack as such areresulted.

In the nerve regeneration-inducing tube of the present invention, a bigeffect is also able to be expected for the adjustment of the degradingrate for bioabsorbency. When a nerve regeneration-inducing tubeconstituted from a biodegradable tubular body and collagen sponge andcoated collagen is embedded in a body, strength of the coated collagenis lost since collagen is firstly degraded. However, when the method ofthe present invention is used, strength of the tubular body is able tobe maintained for a long period since the degrading rate of collagenadhered to the gap between the tubular body fibers is retarded. Further,since the gap between the tubular body fibers is able to be sealed for along period of time whereby it is possible to prevent the invasion ofouter tissues which have a risk of inhibiting the growth of nerve cells.The reason why the degrading rate becomes slow is likely to be due tothe fact that the collagen adhered to the gaps of the tubular bodyfibers has small contact area to the body fluid and to the outertissues.

EXAMPLES

The effect of the nerve regeneration-inducing tube manufactured by themethod of the present invention will be shown below although the presentinvention is not limited thereto. Incidentally, the evaluation of thenerve regeneration-inducing tube obtained in the Examples was done inaccordance with the following methods.

Evaluation Method (1) Pressure Resistance

A load was applied at 100 N/m in a diameter direction from the side of asample in the length of 5 mm as shown in FIG. 1 under the followingmeasuring condition. Then, diameter height (L) in the load direction wasmeasured whereupon a strain rate=(L/L₀)×100 (wherein, L₀ is a diameterheight in the load direction before applying the load) was calculated.Incidentally, the sample was measured for the case of without aging andalso for the case of with aging using a physiological saline solutionfor one, two, three and four week(s).

Measuring Condition

-   -   Temperature: 200° C.; humidity: 65.0%    -   Tester: Tensilon (UTA-1t)    -   Testing speed: 1 mm/min    -   Load cell rating: 5 kgf    -   Sample numbers: N=3

(2) Shape Recovery Property

A sample was compressed until the strain rate became 50% in the diameterdirection from the side of the sample in a length of 5 mm as shown inFIG. 2 under the same measuring condition as in the above (1) pressureresistance Immediately after the compression, the weight was detachedand the sample was allowed to stand for 10 minutes. Then, diameterheight (L₁) in the load direction was measured whereupon a shaperecovery rate=[(L₁−2/L₀)/(2/4)]×100 (wherein, L₀ is a diameter height inthe load direction before applying the load) was calculated.

(3) Anti-Kink Property

As shown in FIG. 3, at the temperature of 20.0° C. and the humidity of65.0%, a sample in the length of 50 mm was bent by hand at the rate ofabout 1 mm/second and the length (L₂ mm) when the kink was generated inthe sample was measured whereupon a limiting curved rate [1−(L₂/50)]×100was calculated. Incidentally, the numbers of the measured sample weremade N=3.

(4) Film Exfoliation Resistance

As shown in FIG. 4, at the temperature of 20.0° C. and the humidity of65.0%, a side of a sample in the length of 5 mm was cut with scissorsand it was confirmed whether the collagen film on the outer surface ofthe sample was able to be exfoliated and separated. Further, the pictureof the outer surface of the sample was taken under an SEM and it wasconfirmed whether partial exfoliation or crack of the film was noted.Incidentally, numbers of the sample to be measured were made N=3.

(5) Cell Invasion Preventing Property and Leakage Resistance

At the temperature of 25.0° C. and the humidity of 60.0%, a 1.0% byweight collagen solution prepared by the method which will be mentionedlater was filled in the inner area of the sample in the length of 5 mm.After that, it was confirmed every ten minutes by naked eye whether thefilled collagen leaked out from the side of the sample and the timeuntil the leakage was confirmed was recorded. When the time until thecollagen solution is completely frozen is taken into consideration, itis necessary that the leakage resistance is not shorter than 2 hours or,at least, not shorter than 1 hour.

Preparation of Collagen Solutions

Into a plastic bottle was placed 392 g of a 0.001 mol/l hydrochloricacid (pH 3), then 8 g of NMP collagen PS (manufactured by Nippon MeatPackers, Inc.) was placed and the mixture was well stirred to dissolvewhereupon a collagen solution in which the final concentration ofcollagen was 2.0% by weight was prepared. This collagen solution wasdiluted with the above hydrochloric acid to prepare collagen solutionsin which the final concentration of collagen was 0.1, 0.2, 0.5, 0.7 and1.0% by weight each.

Measurement of Viscosity of Collagen Solutions

Each of the collagen solutions where collagen concentration was 0.1,0.2, 0.5, 0.7, 1.0 and 2.0% by weight each was stabilized at thetemperature of 10° C. using a constant-temperature vessel in whichcooling water of 10° C. were circulated, then a B type viscometer(product name: Visco Basic plus, manufactured by FUNGILAB, rotor used:L3 spindle, measuring rotation number: 20 rpm, test number: N=3) wasmade to act, the measured values after 3, 4 and 5 minutes from theacting were read and the mean value thereof was adopted as a measuredviscosity. The result is shown in Table 1.

TABLE 1 collagen concentration (% by weight) 0.1 0.2 0.5 0.7 1.0 2.0viscosity (CPS) 17 40 925 2580 7542 25367

Fiber bundle where 28 ultrafine fibers (diameter: about 15 μm)comprising polyglycolic acid were bundled was used as warp and woof andalternately knitted to prepare a cylindrical tubular body of 3 mm innerdiameter and 50 mm length.

Examples 1 to 8, and Comparative Examples 1 and 2

The above collagen solution was uniformly applied for one time onto theouter surface of the above tubular body using a brush made of Teflon(registered trade mark) and air-dried and, after confirming that it wascompletely dried, next application was successively conducted.Concentration and applying times of the collagen solution to be appliedwere in accordance with the description mentioned in the applicationmethod in Table 2 and the applications were successively carried outfrom the solution where the collagen concentration was lower. Aftercompletion of applications of the collagen solutions, a 1.0% by weightcollagen solution was filled into the lumen of the tubular body andfrozen at −40° C. The frozen one was freeze-dried and, after that, athermal cross-linking was carried out in vacuo (not higher than 1 Pa) at140° C. for 24 hours in order to cross-link the collagen molecule andthe resulting one was used as a sample for each of Examples 1 to 8 andComparative Example 1. The sample of Comparative Example 2 was the sameas others except that application of the collagen solution was notcarried out.

Evaluation Result

Pressure resistance, shape recovery property, anti-kink property, filmexfoliation resistence, cell invasion preventing property and leakageresistance were evaluated for the samples of the above Examples 1 to 8and Comparative Examples 1 and 2. The results are shown in Table 2.

TABLE 2 sample No. Example 1 Example 2 Example 3 Example 4 Example 5Example 6 application method 0.1 wt % — — — — — — 0.2 wt % 2 times 2times 2 times 2 times 2 times 2 times 0.5 wt % 1 time 1 time 1 time 1time 1 time 1 time 0.7 wt % — — — — — — 1.0 wt % 17 times 25 times 20times 10 times 5 times 1 time 1.5 wt % — — — — — — strain at 100 N/mwithout aging  5.7%  2.7%  3.9%  3.9%  3.9%  6.5% 1 week 30.2% 29.0%29.0% 19.8% 23.5% 25.8% 2 weeks 43.0% 40.0% 38.8% 47.3% 43.5% 37.0% 3weeks 59.2% 52.7% 55.8% 55.5% 58.0% 56.2% 4 weeks 70.7% 61.8% 69.3%71.2% 76.0% 64.3% shape recovery rate 75.7% 68.5% 70.1% 79.1% 82.8%85.1% limiting curved rate 13.8% 11.0% 14.4% 17.0% 19.4% 20.4% filmseparation absent absent absent absent absent absent exfoliation orcrack absent absent absent absent absent absent observed by SEM timeuntil the filled liquid 180 min 220 min 190 min 130 min 90 min 60 minleaks judgment pressure ∘ ∘∘ ∘∘ ∘∘ ∘∘ ∘ resistance shape recovery ∘ ∘ ∘∘ ∘∘ ∘∘ property anti-kink property ∘ ∘ ∘ ∘∘ ∘∘ ∘∘ film exfoliation ∘ ∘∘ ∘ ∘ ∘ resistance cell invasion ∘∘ ∘∘ ∘∘ ∘∘ ∘ ∘ preventing property andleakage resistance Comparative Comparative sample No. Example 7 Example8 Example 1 Example 2 application method 0.1 wt % 5 times 3 times — —0.2 wt % — — 0 time 0 time 0.5 wt % — 1 time 0 time 0 time 0.7 wt % 25times — — — 1.0 wt % — — 20 times 0 time 1.5 wt % — 10 times — — strainat 100 N/m without aging  5.2%  4.9% 16.2% 48.5% 1 week 30.9% 29.8%35.5% 55.2% 2 weeks 44.1% 42.4% 52.5% 56.8% 3 weeks 60.7% 58.4% 64.5%68.9% 4 weeks 72.5% 69.8% 66.6% 72.5% shape recovery rate 75.6% 74.7%42.6% 81.3% limiting curved rate 14.0% 14.8% 3.6% 23.0% film separationabsent absent present absent exfoliation or crack absent absent presentabsent observed by SEM time until the filled liquid 200 min 190 min 180min 10 min leaks judgment pressure resistance ∘ ∘∘ ∘ Δ shape recovery ∘∘ Δ ∘∘ property anti-kink property ∘ ∘ Δ ∘∘ film exfoliation ∘ ∘ Δ ∘resistance cell invasion ∘∘ ∘∘ ∘∘ Δ preventing property and leakageresistance

It is apparent from the results of Table 2 that the nerveregeneration-inducing tubes manufactured by the method of the presentinvention are excellent in pressure resistance, shape recovery property,anti-kink property, film exfoliation resistance, cell invasionpreventing property and leakage resistance as compared with theconventional ones.

INDUSTRIAL APPLICABILITY

Since the nerve regeneration-inducing tube manufactured by the method ofthe present invention is excellent in the above-mentioned properties, itis excellent in the maintenance of quality during storage or transport,in the handling during the clinical use and in the stability as well assafety after the surgical operation whereby it is quite useful in themedical treatment for nerve regeneration.

1. A method for manufacturing a nerve regeneration-inducing tube,comprising the steps of: applying at least one coat of a first collagensolution to an outer surface of a tubular body, the tubular body beingformed by weaving together a plurality of fibers; applying at least onecoat of a second collagen solution to the outer surface of the tubularbody, the outer surface having been coated with the first collagensolution; filling an inner area of the tubular body with collagen,wherein each of the fibers comprises a biodegradable polymer, andwherein viscosity of the first collagen solution is 5 cps to 200 cps. 2.A method for manufacturing a nerve regeneration-inducing tube,comprising the steps of: applying at least one coat of a first collagensolution to an outer surface of a tubular body, the tubular body beingformed by weaving together a plurality of fibers; applying at least onecoat of a second collagen solution to the outer surface of the tubularbody, the outer surface having been coated with the first collagensolution; applying at least one coat of a third collagen solution to theouter surface of the tubular body, the outer surface having been coatedwith the first collagen solution and the second collagen solution;filling an inner area of the tubular body with collagen, wherein each ofthe fibers comprises a biodegradable polymer, wherein viscosity of thesecond collagen solution is higher than viscosity of the first collagensolution.
 3. The method according to claim 2, wherein viscosity of thethird collagen solution is higher than viscosity of the second collagensolution.
 4. The method according to claim 2, wherein viscosity of thefirst collagen solution is 2 cps to 800 cps, wherein viscosity of thesecond collagen solution is 200 cps to 30,000 cps, and wherein viscosityof the third collagen solution is 200 cps to 30,000 cps.
 5. The methodaccording to claim 4, wherein viscosity of the first collagen solutionis 5 cps to 200 cps, wherein viscosity of the second collagen solutionis 200 cps to 3,000 cps, and wherein viscosity of the third collagensolution is 3,000 cps to 30,000 cps.